Reversibly immortalized hepatocytes and methods of use
a technology of hepatocytes and immortalized cells, which is applied in the field of liver disease and hepatic failure treatment, can solve the problems of human livers, increase the risk of malignant transformation, and severely limit the use of this method for liver failure treatmen
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example 1
Reversible Immortalization of Rat Primary Hepatocytes Using the Cre / Lox System
[0069] In the protocols described in this example, rat primary hepatocytes were immortalized using a recombinant Moloney-based retrovirus containing the gene encoding SV40Tag flanked by loxP sites. Cells were characterized before and after treatment with a recombinant adenovirus capable of transferring the gene encoding the Cre-recombinase to determine whether this approach could produce an hepatocyte cell line that would be useful clinically for transplantation.
Materials and Methods
[0070] Animals. Inbred male Lewis rats (150-250 g) were obtained from Harlan Sprague-Dawley (Indianapolis, Ind.) and maintained in the Animal Resource Facility of the University of Nebraska College of Medicine (Omaha, Nebr.). Animals were maintained on standard laboratory chow on a 12-hour light / dark cycle. Severe combined immunodeficiency (SCID) mice were purchased from the Jackson Laboratory (Bar Harbor, Me.) and maintaine...
example 2
In Vivo Characteristics and Function of loxP / Cre Generated Reversibly Immortalized Rat Hepatocytes
[0097] The functional capacity of 24 individual hepatocyte cell clones that secreted albumin into the culture media was then assessed in an experimental model of inducible hepatic encephalopathy. Total portacaval shunts (PCS) were performed on Lewis rats that were then subjected to ammonium acetate administration. PCS rats that receive exogenous ammonium acetate become hyperammonemic and comatose within 30 minutes (Conjeevaram et al, Hepatology 19: 1245-1250, 1994; Ribiero et al., Hepatology 15:12-18, 1992; Blei et al., Hepatology 19: 1437-1443, 1994). Encephalopathy can also be quantified by means of a coma scale based on evaluation of a variety of behavioral reflexes (Maximum: 15 points indicates normal behavior) (Rigotti et al., Arch Surg. 120: 1290-1295).
[0098] Two weeks after PCS, 3×107 cells from each clone, including C8-B cells, were transplanted by injection into the inferior ...
example 3
Reversibly Immortalized Rat Hepatocyte Transplantation Enhances Liver Function and Survival in Cirrhotic Rats
[0107] Hepatocyte transplantation was shown to be effective in treating liver failure in cirrhotic rats. A determination was made as to whether reversibly immortalized hepatocytes could improve liver function and survival in a model of liver failure physiologically identical to that seen clinically.
[0108] Development of a reproducible model of end-stage liver disease secondary to cirrhosis in rats. None of the classic rodent models for acute liver failure or encephalopathy directly correlate with the physiologic abnormalities associated with the chronic liver dysfunction that is seen in patients. In order to address the role of hepatocyte transplantation in treating decompensated chronic liver disease, liver cirrhosis was induced in rats using phenobarbital (Sigma Chem. Co. St. Louis, Mo.) and carbon tetrachloride (CCl4, Sigma). Lewis rats were given phenobarbital (0.5 g / L)...
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